A method for reducing off-flavors in fruit juice fermented with Lactobacillus plantarum

Abstract

This invention discloses a method for reducing the off-flavor of fermented fruit juice made with *Lactobacillus plantarum*. The method includes: subculturing *Lactobacillus plantarum* under low-oxygen conditions and then inoculating it into fruit juice for fermentation; or pre-acclimating *Lactobacillus plantarum* in a fruit juice gradient culture medium before inoculation and fermentation; or adjusting the initial pH of the fruit juice before inoculation and fermentation. All of the above methods can reduce the off-flavor of fermented fruit juice without inhibiting the growth of the bacterial strain, and are applicable to various fruit juices and various *Lactobacillus plantarum* strains. This invention is simple to operate, highly versatile, and can effectively improve the flavor and quality of fermented fruit juice.

Description

Technical Field

[0001] This invention relates to the field of food microbial fermentation technology, and more particularly to a method for reducing *Lactobacillus plantarum* (Lactobacillus) through pre-fermentation treatment. Lactiplantibacillus plantarum Methods to eliminate off-flavors from fermented fruit juice. Background Technology

[0002] With the increasing demand for healthy foods, fermented fruit juices have become a research hotspot in the functional food field due to their unique flavor characteristics, rich nutritional components, and potential probiotic functions. Among the many microorganisms used for fruit juice fermentation, *Lactobacillus plantarum* (Lactobacillus) is a prominent example. Lactiplantibacillus plantarum With its excellent probiotic properties, outstanding fermentation adaptability, and high substrate conversion efficiency, this bacterium is widely used in the fermentation and processing of various fruit juices. It not only grows well in fruit juice systems and produces metabolites such as organic acids and extracellular polysaccharides, but also effectively enhances the nutritional value and functional activity of the products, thus possessing significant application value in industrial production.

[0003] Fermentation by *Lactobacillus plantarum* not only preserves the characteristic aroma of fruit juice but also generates new volatile flavor compounds through metabolism, giving the product a unique fermented aroma. However, under specific environmental stress or metabolic imbalances, this strain may produce volatile compounds such as 2,3-butanedione, butyric acid, and 4-ethylphenol when metabolizing carbohydrates and nitrogenous compounds. These components are significant contributors to off-flavors, giving fermented fruit juice unpleasant "sour," "rotten," or "buttery" tastes, severely degrading the product's sensory quality. This problem directly restricts consumer acceptance of fermented fruit juice and limits the market promotion and industrial development of related products.

[0004] Currently, strategies for controlling fermentation off-odors mainly include screening for low-odor strains, post-fermentation deodorization treatment, or adding flavor masking agents. However, targeted strain selection is time-consuming and costly, and it is difficult to balance fermentation performance and flavor characteristics; post-fermentation deodorization processes often involve large equipment investments, complex operations, and the potential loss of nutrients; while adding masking agents may contradict the consumer trend of clean labels and even affect the natural properties of the product. Therefore, there is an urgent need in this field for a simple, universally applicable method that can effectively reduce off-odors in Lactobacillus plantarum fermented fruit juice without inhibiting strain growth. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for reducing the off-flavor of fruit juice fermented by Lactobacillus plantarum.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for reducing the off-flavor of fruit juice fermented with Lactobacillus plantarum without significantly reducing the viable count of Lactobacillus plantarum, characterized by comprising at least one pre-fermentation treatment of Lactobacillus plantarum or fruit juice, wherein the pre-fermentation treatment method is as follows: (1) Subculture Lactobacillus plantarum under low oxygen conditions; (2) Pre-acclimatize *Lactobacillus plantarum* in a juice gradient culture medium containing juice; (3) Adjust the initial pH of the juice to a slightly acidic range.

[0007] Preferably, the low-oxygen condition in method (1) is an oxygen volume fraction ≤ 10%. More preferably, the oxygen volume fraction is 0% and 6%.

[0008] Preferably, the juice gradient pre-acclimatization in method (2) includes subculturing in a medium with a gradually increasing juice volume fraction. More preferably, the juice volume fraction is 0%, 20%, 40%, 60%, 80%, or 100%.

[0009] Preferably, the weakly acidic range in method (3) is pH 4.0 to 5.0. More preferably, the initial pH of the juice is adjusted to 4.0 and 5.0.

[0010] Preferably, the fermentation conditions are as follows: after inoculating the fruit juice with *Lactobacillus plantarum*, ferment at 30-45°C for 24-72 hours. More preferably, the fermentation conditions are as follows: ferment at 37°C for 60 hours.

[0011] Preferably, the inoculation amount of *Lactobacillus plantarum* is 5 1 g CFU / mL to 7 1 g CFU / mL. More preferably, the inoculation amount is 7 1 g CFU / mL.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention regulates the metabolic state of *Lactobacillus plantarum* through pre-fermentation treatment methods (hypoxia subculturing, juice gradient pre-acclimatization, or pH adjustment), thereby reducing the production of off-flavor substances during subsequent juice fermentation, effectively improving the flavor quality of fermented juice and enhancing the sensory acceptance of the product. Testing showed that the content of major off-flavor components (such as 2,3-butanedione, butyric acid, 4-ethylphenol, etc.) in fermented juice can be reduced by 30%-99%.

[0013] (2) The methods described in this invention can reduce off-flavors without significantly inhibiting the growth of Lactobacillus plantarum, and the number of viable bacteria at the fermentation endpoint can still be maintained above 7 1g CFU / mL, thus taking into account both the probiotic function and flavor quality of the product.

[0014] (3) The present invention is easy to operate, requires no complicated post-processing deodorization equipment or additional additives, has low production cost, and is easy to promote industrially.

[0015] (4) This invention has broad applicability and is applicable to a variety of fruit juice substrates and a variety of plant lactobacillus strains, providing a flexible and reliable technical option for the large-scale production of fermented fruit juice. Attached Figure Description

[0016] Figure 1 Sensory scores of off-flavors and viable cell counts of grape juice fermented with Lactobacillus plantarum C10 after low-oxygen subculturing. Figure 2 Sensory scores and viable counts of off-flavors from grape juice fermented by Lactobacillus plantarum C10 and Lpgoji after juice gradient pre-acclimation treatment. Figure 3 Sensory scores of off-flavors, pH, and viable count of Lactobacillus plantarum (Lpgoji) fermented mulberry juice after juice gradient pre-acclimation treatment. Figure 4 Sensory scores of off-flavors, pH, and viable count of blueberry juice fermented with Lactobacillus plantarum C10 after juice gradient pre-acclimation treatment. Figure 5 Sensory scores of off-flavors and viable cell counts of grape juice fermented with Lactobacillus plantarum C10 after initial pH adjustment treatment. Figure 6 Sensory scores of off-flavors, pH, and viable count of Lactobacillus plantarum (Lpgoji) fermented mulberry juice after initial pH adjustment treatment. Figure 7 Sensory scores of off-flavors, pH, and viable count of blueberry juice fermented with Lactobacillus plantarum C10 and LB08 after initial pH adjustment treatment. Detailed Implementation This invention provides a method for reducing the off-flavor of fruit juice fermented with *Lactobacillus plantarum*. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0017] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments: Example 1 The specific steps for reducing off-flavors in grape juice fermented with Lactobacillus plantarum using a hypoxic subculturing method are as follows: (1) Hypoxia subculture Lactobacillus plantarum C10 was cultured twice consecutively in environments with 0% and 6% oxygen (v / v), each time at 37°C for 12 h, until the cells reached the logarithmic growth phase. A conventional aerobic subculture served as the control group, with the same subculture number and culture conditions as the experimental group.

[0018] (2) Preparation of seed liquid After passage, the culture medium from each group was collected, centrifuged at 8000 r / min for 1 min, the supernatant was discarded, and the bacterial cell pellet was collected. The bacterial cell pellet was washed twice with sterile physiological saline to remove residual culture medium. The bacterial cells were then resuspended in sterile physiological saline to prepare the fermentation seed culture for later use.

[0019] (3) Fruit juice fermentation Adjust the pH of the grape juice to 4.0±0.1, and inoculate the seed liquid prepared in step (2) into the pH-adjusted grape juice at a bacterial suspension concentration of 7 lg CFU / mL. Let it ferment at 37℃ for 60 h.

[0020] (4) Indicator detection After fermentation, the following tests were performed on each group of samples: pH measurement: The pH value of the fermentation broth was directly measured using a pH meter calibrated with standard buffer solution; Viable cell count was determined by dilution plating method using MRS agar as the medium. After incubation at 37°C for 48 h, the cells were counted, and the results were expressed as 1g CFU / mL. Odor intensity evaluation: An evaluation team composed of trained sensory evaluators (n≥5) used a 9-point scoring system to score the odor intensity of each group of samples. The scoring criteria are as follows: 1 point indicates no odor, and 9 points indicates an extremely strong odor. Odor types were described as negative aroma characteristics that should not be present in fruit juice, such as "stinky rag smell," "sour smell," or "rotten smell." The evaluation was conducted in a standard sensory evaluation room, and the samples were presented with three-digit random codes. The evaluation results were averaged.

[0021] The results are as follows Figure 1 As shown, compared with the conventional aerobic subculturing control group, strain C10 treated with 0% and 6% oxygen subculturing significantly reduced the off-odor sensory score of grape juice fermentation, while maintaining a high viable cell count, both above 8.5 lg CFU / mL. The results indicate that hypoxic subculturing treatment can effectively reduce the off-odor of grape juice fermented by strain C10 without affecting its fermentation activity.

[0022] Further quantitative analysis of the main off-flavor substances in fermented grape juice was conducted, and the results are shown in Table 1. Compared with the control group (CK), the butyric acid content in the 0% oxygen subculture group decreased by approximately 60.5%, the 4-ethylphenol content decreased significantly by 96.4%, and the 2,3-butanedione content decreased by approximately 79.0%. These results indicate that 0% oxygen subculture treatment can significantly reduce the content of the main off-flavor substances in fermented grape juice by strain C10, with a particularly prominent inhibitory effect on 4-ethylphenol. This may be one of the key mechanisms by which hypoxic subculture treatment reduces off-flavors in fermented fruit juice.

[0023] Table 1. Off-flavor substance content (μg / L) of strain C10 after fermentation of grape juice under hypoxic subculturing treatment.

[0024] Example 2 The specific steps for reducing off-flavors in grape juice fermented with Lactobacillus plantarum using a juice gradient pre-acclimation method are as follows: (1) Juice gradient pre-acclimatization Lactobacillus plantarum C10 and Lpgoji were subcultured twice in MRS medium containing 20%, 40%, 60%, 80%, and 100% grape juice, respectively, and cultured at 37°C for 12 h each time until the cells reached the logarithmic growth phase. MRS medium without grape juice served as the control group, with the same subculture number and culture conditions as the experimental group.

[0025] (2) Preparation of seed liquid Same as Example 1.

[0026] (3) Fruit juice fermentation Adjust the pH of the grape juice to 4.0±0.1, and inoculate the seed liquid prepared in step (2) into the pH-adjusted grape juice at a bacterial suspension concentration of 7 lg CFU / mL. Let it ferment at 37℃ for 60 h.

[0027] (4) Indicator detection Same as Example 1.

[0028] The results are as follows Figure 2 As shown, when strain C10 fermented grape juice, the sensory score of off-odor at the fermentation endpoint significantly decreased with increasing grape juice addition during pre-acclimation. Meanwhile, the viable cell count at the fermentation endpoint showed no significant difference compared to the 0% grape juice addition group, remaining between 8.5 and 9.5 1 g CFU / mL. Furthermore, strain LPgoji also showed a trend of reduced off-odor after grape juice pre-acclimation. These results indicate that grape juice pre-acclimation can effectively reduce the off-odor of grape juice fermented by strains C10 and LPgoji without affecting their viable cell count.

[0029] Further quantitative analysis of the main off-flavor substances in grape juice fermented by strain C10 was conducted, and the results are shown in Table 2. With the increase in the proportion of grape juice added during the pre-acclimation process, the content of each off-flavor substance showed a significant decreasing trend. Specifically, the butyric acid content decreased from 32662.714 μg / L in the 0% group to 18192.578 μg / L in the 100% group, a decrease of approximately 44.3%; the 4-ethylphenol content decreased particularly significantly, from 506906.647 μg / L in the 0% group to 6963.520 μg / L in the 100% group, a decrease of up to 98.6%; and the 2,3-butanedione content decreased from 15793.459 μg / L to 4.237 μg / L in the 100% group, almost completely inhibited. The above results indicate that gradient pre-acclimation treatment of grape juice can significantly reduce the content of major off-flavor substances in grape juice fermented by strain C10, and the pre-acclimation effect is positively correlated with the proportion of grape juice added. The inhibitory effect on 4-ethylphenol and 2,3-butanedione is particularly prominent, which may be the key mechanism by which pre-acclimation treatment improves the flavor of fermented juice.

[0030] Table 2. Off-flavor substance content (μg / L) of grape juice fermented by strain C10 after gradient pre-acclimatization treatment.

[0031] Example 3 The following are the specific steps for reducing the off-flavor of mulberry juice fermented with Lactobacillus plantarum using a juice gradient pre-acclimation method: (1) Juice gradient pre-acclimatization Lactobacillus plantarum LPgoji was passaged twice in MRS medium containing 20%, 40%, 60%, 80%, and 100% mulberry juice, and cultured at 37°C for 12 h each time until the cells reached the logarithmic growth phase. MRS medium without mulberry juice served as the control group, with the same passage number and culture conditions as the experimental group.

[0032] (2) Preparation of seed liquid Same as Example 1.

[0033] (3) Fruit juice fermentation Adjust the pH of the mulberry juice to 4.0±0.1, and inoculate the seed liquid prepared in step (2) into the pH-adjusted mulberry juice at a bacterial suspension concentration of 7 lg CFU / mL. Let it ferment at 37℃ for 60 h.

[0034] (4) Indicator detection Same as Example 1.

[0035] The results are as follows Figure 3As shown, the sensory score of off-odor in mulberry juice fermented by strain LPgoji decreased with increasing proportion of mulberry juice added during pre-acclimation. The sensory scores of off-odor in the 40% and 100% mulberry juice addition groups were significantly lower than those in the 0% addition group. There was no significant difference in viable cell count at the fermentation endpoint among the groups. The results indicate that mulberry juice pre-acclimation can reduce the off-odor of fermented mulberry juice without affecting the viable cell count of strain LPgoji.

[0036] Example 4 The following are the specific steps for reducing the off-flavor of blueberry juice fermented with Lactobacillus plantarum using a juice gradient pre-acclimation method: (1) Juice gradient pre-acclimatization Lactobacillus plantarum C10 was passaged twice in MRS medium containing 20%, 40%, 60%, 80%, and 100% blueberry juice, and cultured at 37°C for 12 h each time until the cells reached the logarithmic growth phase. MRS medium without blueberry juice served as the control group, with the same passage number and culture conditions as the experimental group.

[0037] (2) Preparation of seed liquid Same as Example 1.

[0038] (3) Fruit juice fermentation Adjust the pH of the blueberry juice to 4.0±0.1, and inoculate the seed liquid prepared in step (2) into the pH-adjusted blueberry juice at a bacterial suspension concentration of 7 lg CFU / mL. Let it ferment at 37℃ for 60 h.

[0039] (4) Indicator detection Same as Example 1.

[0040] The results are as follows Figure 4 As shown, the sensory scores for off-flavors in fermented blueberry juice were reduced after strain C10 was pre-acclimated to blueberry juice at different proportions. Specifically, the off-flavor score in the 60% addition group only decreased to 3.7 points, while the score in the 100% addition group decreased to 1.3 points. The viable cell count at the fermentation endpoint in the 100% addition group decreased, but remained above 8 1 g CFU / mL, indicating that the pre-acclimation process did not negatively affect the fermentation activity of the strain.

[0041] Example 5 The specific steps for reducing the off-flavor of grape juice fermented with Lactobacillus plantarum using pH adjustment are as follows: (1) Subculture Lactobacillus plantarum C10 was passaged twice in MRS medium, each time at 37°C for 12 h, until the cells reached the logarithmic growth phase.

[0042] (2) Preparation of seed liquid Same as Example 1.

[0043] (3) Fruit juice fermentation Adjust the pH of the grape juice to 4.0±0.1, 4.5±0.1 and 5.0±0.1, and inoculate the seed liquid prepared in step (2) into the grape juice after adjusting the pH at a bacterial suspension concentration of 7 lg CFU / mL, and let it ferment at 37℃ for 60 h.

[0044] (4) Indicator detection Same as Example 1.

[0045] The results are as follows Figure 5 As shown, the sensory score of off-odor in grape juice fermented by strain C10 decreased with increasing pH under different initial pH conditions. The pH 4.0 group had the highest off-odor score, the pH 4.5 group showed a slight decrease, and when the pH was further increased to 5.0, the off-odor score significantly decreased to below 4 points. Meanwhile, the viable cell count at the fermentation endpoint remained above 8.5 lg CFU / mL in all groups. This indicates that the initial pH range of 4.0–5.0 did not significantly inhibit the fermentation activity of strain C10, but it had a significant effect on reducing off-odor.

[0046] Further quantitative analysis of the main off-odor substances in the fermented grape juice was conducted, and the results are shown in Table 3. Compared with the pH 4.0 treatment group, the butyric acid content in the pH 5.0 treatment group decreased by approximately 43.8%; the 4-ethylphenol content decreased significantly to 3395.254 μg / L, a reduction of 99.3%; and the 2,3-butanedione content decreased by approximately 64.5%. These results indicate that adjusting the initial pH of the juice to 5.0 can significantly reduce the content of the main off-odor substances in the fermented grape juice of strain C10, with the most prominent inhibitory effect on 4-ethylphenol, which was almost completely eliminated. This is consistent with the trend of a significant decrease in the off-odor score in the pH 5.0 group in the sensory evaluation results, confirming that adjusting the initial pH is an effective means of controlling off-odors in fermented juice.

[0047] Table 3. Off-flavor substance content (μg / L) of grape juice fermented by strain C10 after initial pH treatment.

[0048] Example 6 The specific steps for reducing the off-flavor of mulberry juice fermented with Lactobacillus plantarum using pH adjustment are as follows: (1) Subculture Lactobacillus plantarum Lpgoji was passaged twice in MRS medium, and cultured at 37°C for 12 h each time until the cells reached the logarithmic growth phase.

[0049] (2) Preparation of seed liquid Same as Example 1.

[0050] (3) Fruit juice fermentation Adjust the pH of the mulberry juice to 4.0±0.1, 4.5±0.1 and 5.0±0.1, and inoculate the seed liquid prepared in step (2) into the mulberry juice after adjusting the pH at a bacterial suspension concentration of 7 lg CFU / mL, and let it ferment at 37℃ for 60 h.

[0051] (4) Indicator detection Same as Example 1.

[0052] The results are as follows Figure 6 As shown, the off-flavor sensory score of mulberry juice fermented by strain Lpgoji under different initial pH conditions showed a continuous downward trend with increasing pH, decreasing from 3.1 points to 1.6 points. The viable cell count at the fermentation endpoint of each group remained stable above 8.2 lg CFU / mL, indicating that Lpgoji maintains good fermentation activity within the pH range of 4.0 to 5.0, and the initial pH has little effect on the survival of the strain.

[0053] Example 7 The following are the specific steps for reducing the off-flavor of blueberry juice fermented with Lactobacillus plantarum using pH adjustment: (1) Subculture Lactobacillus plantarum C10 and LB08 were passaged twice in MRS medium, and cultured at 37°C for 12 hours each time until the cells reached the logarithmic growth phase.

[0054] (2) Preparation of seed liquid Same as Example 1.

[0055] (3) Fruit juice fermentation Adjust the pH of the blueberry juice to 4.0±0.1, 4.5±0.1 and 5.0±0.1, and inoculate the seed liquid prepared in step (2) into the blueberry juice after adjusting the pH at a bacterial suspension concentration of 7 lg CFU / mL, and let it ferment at 37℃ for 60 h.

[0056] (4) Indicator detection Same as Example 1.

[0057] The results are as follows Figure 7 As shown, the sensory score for off-flavor in blueberry juice fermented by strain C10 under different initial pH conditions showed a trend of first decreasing and then increasing. The highest score was 4.4 points in the pH 4.0 group, which decreased to 3.5 points in the pH 4.5 group, and rebounded to 4.2 points in the pH 5.0 group. The viable cell count at the fermentation endpoint was greater than 8.6 lg CFU / mL in all groups.

[0058] The odor sensory score of strain LB08 showed a continuous decreasing trend with increasing initial pH. The highest score was 4.3 points in the pH 4.0 group, which dropped to 4.0 points in the pH 4.5 group, and further decreased to 3.5 points in the pH 5.0 group. The viable cell count at the fermentation endpoint was greater than 8 1 g CFU / mL in all groups, and increased slightly with increasing pH.

[0059] In summary, through the verification of the above embodiments, the present invention provides three effective methods for reducing the off-flavor of fruit juice fermented by *Lactobacillus plantarum*, as follows: (1) Subculture Lactobacillus plantarum under low oxygen conditions; (2) Pre-acclimatize *Lactobacillus plantarum* in a juice gradient culture medium containing juice; (3) Adjust the initial pH of the juice to a slightly acidic range.

[0060] The above three methods can be used individually or in combination as needed for actual production. Through multiple examples of fermenting different fruit juices with different strains, it has been verified that all of the above methods can effectively reduce undesirable aromas such as "stinky rag smell," "sour smell," and "rotten smell" produced during the fermentation of fruit juice by *Lactobacillus plantarum*, while maintaining a high level of viable bacteria in the fermented fruit juice, thus meeting the quality requirements of fermented fruit juice products.

[0061] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for reducing the off-flavor of fruit juice fermented with Lactobacillus plantarum, characterized in that, This includes at least one pre-fermentation treatment of *Lactobacillus plantarum* or fruit juice, wherein the pre-fermentation treatment method is as follows: (1) Subculture Lactobacillus plantarum under low oxygen conditions; (2) Pre-acclimatize *Lactobacillus plantarum* in a juice gradient culture medium containing juice; (3) Adjust the initial pH of the juice to a slightly acidic range.

2. The method according to claim 1, characterized in that, In method (1), the low-oxygen condition is that the oxygen volume fraction is ≤10% and the number of subcultures is 2.

3. The method according to claim 1, characterized in that, In method (2), the juice gradient pre-acclimatization includes subculturing in a medium with a gradually increasing juice volume fraction, and the number of subculturing times is 2.

4. The method according to claim 2 or 3, characterized in that, The passage includes: inoculating *Lactobacillus plantarum* into a culture medium according to the method of claim 2 or 3, activating it at 35-40°C, passaged every 8-15 h, and after culturing for 30-40 h, removing the culture medium, resuspending the bacterial cells with physiological saline to obtain a seed culture.

5. The method according to claim 1, characterized in that, In method (3), the weak acid range is pH 4.0~5.

0.

6. The method according to claim 1, characterized in that, The fermentation conditions are as follows: after inoculating the fruit juice with Lactobacillus plantarum, ferment at 30-45°C for 24-72 hours.

7. The method according to claim 6, characterized in that, The inoculation amount of *Lactobacillus plantarum* was 5 1g CFU / mL to 7 1g CFU / mL.

8. The method according to claim 6, characterized in that, The juice is the juice whose initial pH has been adjusted by method (3).

9. The method according to claim 1, characterized in that, The pre-fermentation treatment did not significantly reduce the viable count of *Lactobacillus plantarum*, and the viable count at the fermentation endpoint was not less than 7 1 g CFU / mL.

10. The method according to claim 1, characterized in that, After the aforementioned pre-fermentation treatment, the off-flavor components (2,3-butanedione, butyric acid, and 4-ethylphenol) in the fermented fruit juice are reduced by 30%-99%.

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